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. 2021 May 10;14(9):2461.
doi: 10.3390/ma14092461.

Bioactive Regeneration Potential of the Newly Developed Uncalcined/Unsintered Hydroxyapatite and Poly-l-Lactide-Co-Glycolide Biomaterial in Maxillofacial Reconstructive Surgery: An In Vivo Preliminary Study

Shinji Ishizuka  1 , Quang Ngoc Dong  1 , Huy Xuan Ngo  1 , Yunpeng Bai  1 , Jingjing Sha  1 , Erina Toda  1 , Tatsuo Okui  1 , Takahiro Kanno  1
Affiliations

Bioactive Regeneration Potential of the Newly Developed Uncalcined/Unsintered Hydroxyapatite and Poly-l-Lactide-Co-Glycolide Biomaterial in Maxillofacial Reconstructive Surgery: An In Vivo Preliminary Study

Shinji Ishizuka et al. Materials (Basel). .

Abstract

Uncalcined/unsintered hydroxyapatite (HA) and poly-l-lactide-co-glycolide (u-HA/PLLA/PGA) are novel bioresorbable bioactive materials with bone regeneration characteristics and have been used to treat mandibular defects in a rat model. However, the bone regenerative interaction with the periosteum, the inflammatory response, and the degradation of this material have not been examined. In this study, we used a rat mandible model to compare the above features in u-HA/PLLA/PGA and uncalcined/unsintered HA and poly-l-lactic acid (u-HA/PLLA). We divided 11 male Sprague-Dawley rats into 3- and 16-week groups. In each group, we assessed the characteristics of a u-HA/PLLA/PGA sheet covering the right mandibular angle and a u-HA/PLLA sheet covering the left mandibular angle in three rats each, and one rat was used as a sham control. The remaining three rats in the 16-week group were used for a degradation assessment and received both sheets of material as in the material assessment subgroup. At 3 and 16 weeks after surgery, the rats were sacrificed, and mandible specimens were subjected to micro-computed tomography, histological analysis, and immunohistochemical staining. The results indicated that the interaction between the periosteum and u-HA/PLLA/PGA material produced significantly more new bone regeneration with a lower inflammatory response and a faster resorption rate compared to u-HA/PLLA alone. These findings may indicate that this new biomaterial has ideal potential in treating maxillofacial defects of the midface and orbital regions.

Keywords: CD68; Runx2; bone regeneration; osteocalcin; osteoconductivity; periostin; poly-l-lactic acid; poly-l-lactide-co-glycolide; uncalcined/unsintered hydroxyapatite.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Surgical procedure. (A) u-HA/PLLA sheet (left) and u-HA/PLLA/PGA sheet (right) with the same dimensions: 5 mm length × 5 mm width × 0.3 mm thickness. (B) The mandibular angle area was exposed on the right side. (C) Fixation of the reconstruction material using a hemoclip. The black vertical lines in (D,E) indicate the sites at which samples were collected for analysis. u-HA/PLLA material was applied on the left side and u-HA/PLLA/PGA material on the right side. (F) Schematic coronal view of one specimen.
Figure 2
Figure 2
Experimental groups.
Figure 3
Figure 3
Micro-CT assessment. (A) Image of the initial micro-CT data. (B) Illustration of a slice image in which the outer bone was easily detected after reorienting. (C) Illustrated overview of one specimen. The area of newly formed bone on the buccal side of the material sheet was measured in each slice using the Fiji “ROI Manager” tool.
Figure 4
Figure 4
Example of ROI selection for areas containing new bone (anti-OCN IHC staining). (A) Original image with overlay of the selected ROI. *, selected area. (B) Hematoxylin-stained image separated from the original image. (C) DAB-stained image separated from the original image. (D) Superimposition of the saved ROI onto the DAB-stained image. *, selected area.
Figure 5
Figure 5
Micro-CT images of rat hemi-mandibles. The u-HA/PLLA/PGA is not visible due to the low proportion of u-HA. 3D view of the (A) u-HA/PLLA/PGA subgroup, (B) u-HA/PLLA subgroup, and (C) sham control group. Scale bar: 5 mm.
Figure 6
Figure 6
Volume of new bone, as determined by micro-CT evaluation, in the u-HA/PLLA/PGA and u-HA/PLLA subgroups. * p < 0.05.
Figure 7
Figure 7
Hematoxylin-and-eosin-stained sections from the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. Images of each subgroup were taken at 1.25×, 4×, and 20× magnification (from left to right). Images of the sham control group were taken at 1.25× magnification. (A) u-HA/PLLA/PGA subgroup. (B) Sham control group. (C) u-HA/PLLA subgroup. Scale bar: 1 mm (1.25× magnification), 500 μm (4× magnification), and 100 μm (20× magnification).
Figure 8
Figure 8
Runx2 expression in the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. Images in each subgroup were taken at 1.25×, 4×, and 20× magnification (from left to right). Images of the sham control group were taken at 20× magnification. (A) u-HA/PLLA/PGA subgroup. (B) Sham control group. (C) u-HA/PLLA subgroup. Scale bar: 1 mm (1.25× magnification), 500 μm (4× magnification), and 100 μm (20× magnification).
Figure 9
Figure 9
Runx2 labeling index in the u-HA/PLLA/PGA and u-HA/PLLA subgroups. * p < 0.05.
Figure 10
Figure 10
OCN expression in the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. All images were taken at 4× magnification. Scale bar: 500 μm.
Figure 11
Figure 11
Digital H-scores based on IHC staining using an anti-OCN antibody in the u-HA/PLLA/PGA and u-HA/PLLA subgroups.
Figure 12
Figure 12
Periostin expression in the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. Images in each subgroup were taken at 1.25× and 20× magnification (from left to right). Images of the sham control group were taken at 1.25× magnification. (A) u-HA/PLLA/PGA subgroup. (B) Sham control group. (C) u-HA/PLLA subgroup. Scale bar: 1 mm (1.25× magnification) and 100 μm (20× magnification).
Figure 12
Figure 12
Periostin expression in the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. Images in each subgroup were taken at 1.25× and 20× magnification (from left to right). Images of the sham control group were taken at 1.25× magnification. (A) u-HA/PLLA/PGA subgroup. (B) Sham control group. (C) u-HA/PLLA subgroup. Scale bar: 1 mm (1.25× magnification) and 100 μm (20× magnification).
Figure 13
Figure 13
Digital H-scores based on IHC staining using an anti-periostin antibody in the u-HA/PLLA/PGA and u-HA/PLLA subgroups. * p < 0.05.
Figure 14
Figure 14
CD68 expression in the u-HA/PLLA/PGA subgroup, u-HA/PLLA subgroup, and sham control. Images in each subgroup were taken at 4× and 20× magnification (from left to right). Images of the sham control group were taken at 1.25× magnification. (A) u-HA/PLLA/PGA subgroup. (B) Sham control group. (C) u-HA/PLLA subgroup. Scale bar: 1 mm (1.25× magnification image), 500 μm (4× magnification image), and 100 μm (20× magnification image).
Figure 15
Figure 15
Labeling index based on IHC staining using an anti-CD68 antibody in the u-HA/PLLA/PGA and u-HA/PLLA subgroups.
Figure 16
Figure 16
Retention rates of u-HA/PLLA/PGA and u-HA/PLLA. * p < 0.05.
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